Soybean raffinose synthase and a method for producing raffinose

ABSTRACT

Raffinose is produced by allowing a raffinose synthase having the following properties to act on sucrose and galactinol:
         (1) action and substrate specificity: produces raffinose from sucrose and galactinol;   (2) optimum pH: about 6 to 8;   (3) optimum temperature: about 35 to 40° C.;   (4) molecular weight:   (i) about 75 kDa to 95 kDa estimated by gel filtration chromatography;   (ii) about 90 kDa to 100 kDa estimated by polyacrylamide gel electrophoresis (Native PAGE); and   (iii) about 90 kDa to 100 kDa estimated by SDS-polyacrylamide gel electrophoresis under a reduced condition (SDS-PAGE); and   (5) inhibition: inhibited by iodoacetamide, N-ethylmaleimide, and myo-inositol.

This application is a Divisional of U.S. application Ser. No. 09/425,055filed on Oct. 22, 1999, now U.S. Pat. No. 6,891,084, which is aContinuation of International Application PCT/JP97/03879 filed on Oct.24, 1997, which is a Continuation-in-Part of U.S. application Ser. No.08/846,234 filed on Apr. 28, 1997, now U.S. Pat. No. 6,166,292.

TECHNICAL FIELD

The present invention relates to a raffinose synthase, a method forraffinose synthesis using the raffinose synthase or a cell-free extractcontaining the raffinose synthase, a DNA coding for the raffinosesynthase, and methods for its in plants. Raffinose is utilized in avariety of fields, as a food material having an activity to proliferateBifidobacterium, or as a pharmaceutical to be used, for example, forsolutions of organ preservation.

BACKGROUND ART

Raffinose is one of raffinose family oligosaccharides, in whichgalactose is connected to glucosyl group of sucrose via α-1,6 linkage.The raffinose family oligosaccharides include, for example, stachyosecontaining two connected galactose residues, and verbascose containingthree connected galactose residues, in addition to raffinose. Theseoligosaccharides are widely distributed in plants, for example, seeds ofvarious plants such as beans, rapeseed, and cottonseed containing theseoligosaccharides as reserve carbohydrates; plants belonging toCucurbitaceae such as cucumber and melon containing these sugars astranslocation sugars; and sugar beet (Beta vulgaris) and rosette leaveshaving acquired cold resistance.

The raffinose family oligosaccharides are biosynthesized as follows.UDP-galactose+myo-inositol−galactinol+UDP   (a)galactinol+sucrose−raffinose+myo-inositol   (b)galactinol+raffinose−stachyose+myo-inositol   (c)

The reactions are catalyzed by (a) galactinol synthase (GS: EC2.4.1.123), (b) raffinose synthase (RS: EC 2.4.1.82), and (c) stachyosesynthase (STS: EC 2.4.1.67), respectively.

At present, raffinose is extracted from sugar beet, and it is separatedand purified in the sucrose purification process. However, since crystalformation of sucrose is deteriorated by raffinose, sugar beet has beensubjected to breeding and improvement with the aim of decreasing theraffinose content. As a result, the raffinose content in sugar beet nowhas a low value of 0.03% to 0.16% (Enzyme Microb. Technol., Vol. 4, May,130-135 (1982)). Therefore, it is not easy to efficiently obtainraffinose from sugar beet having such a low raffinose content.

As described above, raffinose is contained in mature seeds ofLeguminosae plants represented by soybean and in sugar beet andCucurbitaceae plants such as cucumber. Mature seed of soybean contains,as soybean oligosaccharides, sucrose (content: about 5%), stachyose(content: about 4%), and raffinose (content: about 1%). The soybeanoligosaccharides are recovered in a fraction obtained by deproteinizingdefatted soybean, and they are utilized, for example, for functionalfood products after concentration. However, raffinose occupies aproportion of 10% of the whole oligosaccharides, and hence raffinoseexists in a small amount.

On the other hand, a method for enzymatically synthesizing raffinose hasbeen reported (Trends in Glycoscience and Glycotechnology, 7.34, 149-158(1995)). This method comprises the steps of synthesizing galactobiose inaccordance with a condensation reaction catalyzed by α-galactosidase,and transferring galactosyl group to sucrose by using the galactobioseas a galactosyl group donor in accordance with a galactosyl transferreaction to synthesize raffinose. However, in this reaction, 350 g ofgalactobiose is synthesized from 1.9 kg of lactose hydrolysate, and 100g of raffinose is obtained from 190 g of galactobiose and 760 g ofsucrose. Therefore, the yield of produced raffinose is low, and hencethis synthesis method is not efficient.

Besides the foregoing methods, a method is also conceivable in which aplant having a high raffinose content may be bred by means oftransformation for genes for enzymes involved in the biosynthesissystem. For example, Kerr et al. have cloned a gene for galactinolsynthase, and transformed rapeseed therewith (WO 93/02196). As a result,the GS activity was increased, however, the content of the raffinosefamily oligosaccharides was unwillingly decreased. It was impossible toachieve the object to enhance the biosynthesis of the raffinose familyoligosaccharides by introducing the galactinol synthase gene. Therefore,there has not been provided a method for increasing the content of theraffinose family oligosaccharides in plant.

On the other hand, it is also demanded to decrease the raffinose familyoligosaccharides. As described above, the raffinose familyoligosaccharides are widely distributed over plants including, seeds ofvarious plants such as beans, for example, soybean, rapeseed, andcottonseed containing these oligosaccharides as storage carbohydrates;Cucurbitaceae plants such as cucumber and melon containing theseoligosaccharides as translocation sugars; and sugar beet and rosetteleaves having acquired cold resistance. Meals obtained after extractionof oil, for example, from soybean, rapeseed, and cotton contain theraffinose family oligosaccharides. Almost all of the meals are utilizedas feed. However, human and animals, which do not have α-galactosidase,cannot directly digest the raffinose family oligosaccharides. It isknown that the raffinose family oligosaccharides lower the metabolicenergy efficiency of feed due to, for example, assimilation of theraffinose family oligosaccharides by enteric bacteria to cause gasproduction. It has been reported that removal of raffinose familyoligosaccharides from soybean meal results in a large increase in themetabolizable energy for broiler chickens (Coon, “Proceeding SoybeanUtilization Alternatives”, University of Minnesota, 203-211 (1989)). Inview of the foregoing facts, it is desired to develop the plants such assoybean, rapeseed, and cottonseed in which the raffinose familyoligosaccharides are decreased.

Such plants have been subjected to breeding to increase the amount ofoil. Photosynthetic products are distributed among oils, proteins, andcarbohydrates including the raffinose family oligosaccharides. It hasbeen reported for soybean that a reverse correlation exists between theamount of oils and the amount of carbohydrates. It is expected that thecontent of oils can be increased in a soybean plant having the samephotosynthetic ability as those possessed by others, by decreasing theproduction of the raffinose family oligosaccharides.

Based on a viewpoint as described above, Kerr et al. have reporteddevelopment of soybean varieties with a low content of the raffinosefamily oligosaccharides, by means of breeding based on mating andselection, in which the raffinose family oligosaccharides are lowered byan amount of 80% to 90% (WO 93/00742). However, this technique concernscreation of soybean variety, which cannot be applied to other varioussoybean varieties developed in response to, for example, aptitude forcultivation and resistance to disease. This technique cannot beuniversally applied to various plants as well.

It is known that raffinose, which is contained, for example, in sugarbeet and sugar cane, lowers crystal formation of sugar or sucrose.Therefore, it is possible to expect that if no raffinose is produced,the production efficiency of sugar may be improved in such a plant.However, no sugar beet has been created, which contains no raffinose.

As described above, the raffinose synthase, which has been hithertopurified, has been confirmed only as an enzyme activity, and no entityof the enzyme has been identified. The confirmed activity is low, and ithas been desired to obtain a raffinose synthase having a high activity.The conventional method for producing raffinose provides a low yield,and hence it has been desired to develop an efficient method forproducing raffinose. On the other hand, it is also desired to breed aplant in which the raffinose family oligosaccharides are decreased.

DISCLOSURE OF THE INVENTION

The present invention has been made taking the foregoing viewpoints intoconsideration, and an object of the present invention is to obtain araffinose synthase having a high activity and a DNA encoding theraffinose synthase, and provide an efficient method for enzymaticallysynthesizing raffinose, and a method for utilizing the DNA encoding theraffinose synthase in plants.

As a result of diligent investigations in order to achieve the objectdescribed above, the present inventors have succeeded in purifying araffinose synthase from cucumber. Further diligent investigations havebeen made by the present inventors in order to clone a gene coding forthe raffinose synthase. As a result, a DNA fragment specific to a genefor the raffinose synthase has been obtained by chemically synthesizingsingle strand DNAs on the basis of nucleotide sequences deduced fromamino acid sequences of peptide fragments of the cucumber raffinosesynthase, and performing PCR by using the single strand synthetic DNAsas primers and using cDNAs prepared from poly(A)⁺RNA extracted fromcucumber as templates. Further, the raffinose synthase gene has beenisolated by adopting a method in which hybridization is performed for acDNA library originating from cucumber by using the DNA fragment as aprobe. Also, diligent investigations in order to clone a raffinosesynthase gene of soybean origin have been made based on informationabout the raffinose synthase gene of cucumber origin. As a result, theraffinose synthase gene of soybean origin has been isolated. A chimericgene having a regulatory region expressible in plants has been preparedby using a fragment of the isolated raffinose synthase gene to transforma plant. Further, a plant in which the raffinose family oligosaccharidesare decreased due to the introduced raffinose synthase gene, has beencreated.

Namely, the present invention provides a raffinose synthase having anactivity to produce raffinose from sucrose and galactinol.

Preferably, the present invention provides a raffinose synthase which isa protein specified by the following (A), (B), (C) or (D):

(A) a protein which has an amino acid sequence shown in SEQ ID NO: 5 inSequence Listing;

(B) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 5 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol;

(C) a protein which has an amino acid sequence shown in SEQ ID NO: 24 inSequence Listing; or

(D) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 24 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol.

Also, the present invention provides a raffinose synthase which has thefollowing properties:

(1) action and substrate specificity: produces raffinose from sucroseand galactinol;

(2) optimum pH: about 6 to 8;

(3) optimum temperature: about 35 to 40° C.;

(4) molecular weight:

(i) about 75 kDa to 95 kDa estimated by gel filtration chromatography;

(ii) about 90 kDa to 100 kDa estimated by polyacrylamide gelelectrophoresis (Native PAGE); and

(iii) about 90 kDa to 100 kDa estimated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) under a reduced condition; and

(5) inhibition: inhibited by iodoacetamide, N-ethylmaleimide, andmyo-inositol.

In an embodiment of the foregoing raffinose synthase provided by thepresent invention, the raffinose synthase has an amino acid sequenceincluding amino acid sequences shown in SEQ ID NOs: 28 to 30 in SequenceListing.

The present invention also provides a raffinose synthase which is aprotein specified by the following (C) or (D):

(C) a protein which has an amino acid sequence shown in SEQ ID NO: 24 inSequence Listing; or

(D) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 24 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol.

In another aspect of the present invention, there is provided a methodfor producing raffinose, comprising the step of allowing the foregoingraffinose synthase to act on sucrose and galactinol to produceraffinose.

In still another aspect of the present invention, there are provided aDNA encoding the raffinose synthase, and, in particular, a DNA codingfor a protein specified by the following (A), (B), (C) or (D):

(A) a protein which has an amino acid sequence shown in SEQ ID NO: 5 inSequence Listing;

(B) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 5 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol;

(C) a protein which has an amino acid sequence shown in SEQ ID NO: 24 inSequence Listing; or

(D) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 24 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol.

In an embodiment of the foregoing DNA of the present invention, there isprovided a DNA specified by the following (a), (b), (c) or (d):

(a) a DNA which includes a nucleotide sequence comprising at leastnucleotide residues of nucleotide numbers 56 to 2407 in a nucleotidesequence shown in SEQ ID NO: 4 in Sequence Listing;

(b) a DNA which hybridizes under stringent conditions with thenucleotide sequence comprising at least nucleotide residues ofnucleotide numbers 56 to 2407 in the nucleotide sequence shown in SEQ IDNO: 4 in Sequence Listing, and which codes for a protein having anactivity to produce raffinose from sucrose and galactinol;

(c) a DNA which includes a nucleotide sequence comprising at leastnucleotide residues of nucleotide numbers 156 to 2405 in a nucleotidesequence shown in SEQ ID NO: 23 in Sequence Listing; or

(d) a DNA which hybridizes under stringent conditions with thenucleotide sequence comprising at least nucleotide residues ofnucleotide numbers 156 to 2405 in the nucleotide sequence shown in SEQID NO: 23 in Sequence Listing, and which codes for a protein having anactivity to produce raffinose from sucrose and galactinol.

In still another aspect of the present invention, there is provided aDNA useful for expression of an antisense RNA or a sense RNA of theraffinose synthase, namely, a DNA specified by the following (e) or (f):

(e) a DNA which hybridizes under stringent conditions with a nucleotidesequence comprising at least nucleotide residues of nucleotide numbers56 to 2407 in a nucleotide sequence shown in SEQ ID NO: 4 in SequenceListing, or a complementary nucleotide sequence thereof; or

(f) a DNA which hybridizes under stringent conditions with a nucleotidesequence comprising at least nucleotide residues of nucleotide numbers156 to 2405 in a nucleotide sequence shown in SEQ ID NO: 23 in SequenceListing, or a complementary nucleotide sequence thereof.

In still another aspect of the present invention, there are provided achimeric-gene comprising the raffinose synthase gene or a part thereof,and a transcription regulatory region expressible in plant cells, and aplant transformed with the chimeric gene.

In still another aspect of the present invention, there is provided amethod for changing a content of raffinose family oligosaccharides in aplant, comprising the steps of transforming the plant with the chimericgene, and allowing the gene to be expressed in the plant.

In the following description, the raffinose synthase having theproperties described in the foregoing (1) to (5), or the raffinosesynthase specified as the protein defined in the foregoing (A), (B), (C)and (D) is simply referred to as “raffinose synthase” in some cases. TheDNA encoding raffinose synthase, or the DNA encoding raffinose synthaseand including non-translating regions is referred to as “raffinosesynthase gene” in some cases.

The present invention will be explained in detail below.

<1> Raffinose Synthase of the Present Invention

The raffinose synthase of the present invention may be a proteinspecified by the following (A), (B), (C) or (D):

(A) a protein which has an amino acid sequence shown in SEQ ID NO: 5 inSequence Listing;

(B) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 5 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol;

(C) a protein which has an amino acid sequence shown in SEQ ID NO: 24 inSequence Listing; or

(D) a protein which comprises an amino acid sequence includingsubstitution, deletion, insertion, addition, or inversion of one orseveral residues of amino acids in the amino acid sequence shown in SEQID NO: 24 in Sequence Listing, and which has an activity to produceraffinose from sucrose and galactinol.

The raffinose synthase of the present invention includes that having thefollowing properties:

(1) action and substrate specificity: produces raffinose from sucroseand galactinol;

(2) optimum pH: about 6 to 8;

(3) optimum temperature: about 35 to 40° C.;

(4) molecular weight:

(i) about 75 kDa to 95 kDa estimated by gel filtration chromatography;

(ii) about 90 kDa to 100 kDa estimated by polyacrylamide gelelectrophoresis (Native PAGE); and

(iii) about 90 kDa to 100 kDa estimated by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) under a reduced condition; and

(5) inhibition: inhibited by iodoacetamide, N-ethylmaleimide, andmyo-inositol.

The raffinose synthase having the foregoing properties has been isolatedand purified from leaves of cucumber, and has been identified for thefirst time by the present inventors. As demonstrated in Examplesdescribed later, the raffinose synthase of cucumber origin includes theamino acid residues shown in SEQ ID NOs: 1 to 3 or SEQ ID NOs: 28 to 30in Sequence Listing, in the amino acid sequence of the enzyme protein.An entire amino acid sequence of the raffinose synthase is shown in SEQID NO: 5.

The raffinose synthase is obtainable from plants belonging toCucurbitaceae, for example, plants such as melon (Cucumis melo) andcucumber (Cucumis sativus). Especially, the raffinose synthase iscontained in a large amount in leaves of these plants, especially intissues of leaf vein portions and seeds.

Next, the method for producing the raffinose synthase of the presentinvention will be explained in accordance with an illustrative methodfor isolating and purifying the raffinose synthase from cucumber.

Leaf vein portions are collected from leaves of cucumber obtained 6 to10 weeks after planting, and ground with liquid nitrogen by, forexample, a mortar. Then, a buffer is added thereto to extract proteins.During this process, it is allowable to add a substance to prevent theraffinose synthase from degradation and inactivation, for example, aprotease inhibitor such as PMSF (phenylmethane-sulfonyl fluoride), orpolyclarl AT (produced by Serva). Insoluble matters are removed from anobtained extract solution by means of filtration and centrifugation toobtain a crude extract solution.

The crude extract solution thus obtained is subjected to fractionationbased on combination of ordinary methods for purifying proteins,including, for example, anion exchange chromatography, hydroxyapatitechromatography, gel filtration, and salting out. Thus the raffinosesynthase can be purified.

Anion exchange chromatography can be performed, for example, by using acolumn charged with a strongly basic anion exchanger such as HiTrap Q(produced by Pharmacia), or a weakly basic anion exchanger such asDEAE-TOYOPEARL (produced by Tosoh Corp.). The extract solutioncontaining the raffinose synthase is allowed to pass through the columnso that the enzyme is adsorbed to the column. After washing the column,the enzyme is eluted by using a buffer having a high salt concentration.During this process, the salt concentration may be increased in astepwise manner, or the concentration gradient may be applied. Forexample, when the HiTrap Q column is used, the raffinose synthaseactivity adsorbed to the column is eluted by NaCl at about 0.3 M. Aneluting solution to give an NaCl concentration gradient of 0.05 M to0.35 M is preferably used for DEAE-TOYOPEARL. An eluting solution togive a phosphate concentration gradient of 0.01 M to 0.3 M is preferablyused for hydroxyapatite chromatography.

The order of the foregoing operations is not specifically limited. Eachof the operations may be repeated two or more times. It is desirable toexchange a sample solution with an appropriate buffer by means ofdialysis or the like before the sample solution is allowed to passthrough each column. The sample solution may be concentrated at eachstage.

At each stage of the purification, it is preferable that the raffinosesynthase activity contained in each of fractionated fractions ismeasured so that fractions having high activities are collected to beused in the next stage. The method for measuring the raffinose synthaseactivity is exemplified by a method using radioisotope as reported, forexample, by Lehle, H. et al. (Eur. J. Biochem., 38, 103-110 (1973)). Asa modified method thereof, the reaction temperature and the substrateconcentration may be changed. For example, 10 μl of an enzyme solutionis added to a reaction solution containing, at final concentrations, 10mM ¹⁴C-sucrose, 20 mM galactinol, 25 mM HEPES(2-(4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid)-NaOH, pH 7.0,0.5 mM DTT (dithiothreitol) to give a volume of 50 μl. The solution isincubated at 32° C. for 1 hour to perform the reaction. The reaction isstopped by adding 200 μl of ethanol and heating the solution at 95° C.for 30 seconds. The reaction solution is centrifuged to obtain asupernatant. An aliquot of the supernatant is spotted on Whatman 3MMfilter paper, and developed with n-propanol:ethyl acetate:water=4:1:2.Incorporation of ¹⁴C into raffinose is investigated, which is regardedto be the raffinose synthase activity (nmol/hour).

The present inventors have developed a method for measuring theraffinose synthase activity in place of the foregoing method. Namely,the raffinose synthase activity is measured by quantitativelydetermining raffinose produced by the raffinose synthesis reaction, bymeans of HPLC (high-performance liquid chromatography). According tothis method, the activity can be measured conveniently and quickly ascompared with the method of Lehle, H. et al. This method is especiallypreferable to detect active fractions during the purification operation.This method will be explained below.

For the raffinose synthesis reaction, to a reaction solution prepared tohave a composition having the following final concentrations, 10 to 50μl of a raffinose synthase solution is added to give a volume of 100 μl,followed by performing the reaction at 32° C. for 60 minutes.

[Composition of Reaction Solution (Final Concentration)]

2.5 mM sucrose   5 mM galactinol   5 mM DTT  20 mM Tris-HCl buffer (pH7.0)

After performing the reaction as described above, the reaction isstopped by adding to the reaction solution, ethanol in a volume fourtimes the volume of the reaction solution and heating the solution at95° C. for 30 seconds. The obtained solution is centrifuged to obtain asupernatant and the supernatant is then dried up under a reducedpressure. After that, an obtained residue is dissolved in distilledwater. Raffinose in the reaction product is quantitatively determined byusing HPLC to estimate the raffinose synthase activity. HPLC can beperformed by using, for example, Sugar Analysis System DX500 (CarboPacPA1 column, pulsed amperometry detector (produced by Dionecs)).

FIG. 1 shows a result of measurement performed in accordance with themethod described above, for the amount of raffinose produced when thereaction time was changed. As seen from FIG. 1, this method makes itpossible to conveniently measure the raffinose synthase activity withexcellent linearity.

The degree of purification of the purified raffinose synthase can beconfirmed, and the molecular weight can be measured, by means of, forexample, gel electrophoresis and gel filtration chromatography.Enzymatic properties can be investigated by measuring the enzymeactivity while changing the reaction temperature or the reaction pH, orby measuring the remaining enzyme activity after adding, to the reactionsolution, various enzyme inhibitors, metal ions or the like. The stablepH range and the stable temperature range can be investigated bymeasuring the enzyme activity after exposing the raffinose synthase tovarious pH conditions and temperature conditions for a certain period oftime respectively.

The properties of the raffinose synthase described above have beendetermined in accordance with procedures as described above. However, itshould be noted that different results may be obtained depending onmeasurement conditions. For example, the measurement for the molecularweight based on the use of gel filtration chromatography is affected bythe type of the gel filtration carrier and the buffer, and the molecularweight marker to be used. The enzyme activity differs depending on thetype of the buffer and the salt concentration in many cases even whenthe measurement is performed at an identical pH. Therefore, uponidentification for the raffinose synthase, it is preferable to performcomprehensive investigation without being bound to only measurement forindividual properties.

The raffinose synthase of the present invention can be obtained byperforming the isolation and purification from cucumber as describedabove. Alternatively, the raffinose synthase of the present inventioncan be produced by introducing, into an appropriate host, a DNA codingfor the raffinose synthase originating from cucumber, soybean or anotherplant as described later, and making expression thereof, in accordancewith ordinary methods used for fermentative production of heterogeneousproteins.

Those assumed as the host for expression of the raffinose synthase geneinclude various procaryotic cells represented by Escherichia coli, andvarious eucaryotic cells represented by Saccharomyces cerevisiae.However, it is desirable to use plant cells, especially cellsoriginating from plants such as tobacco, cucumber, and Arabidopsisthaliana.

The recombinant plasmid used for transformation can be prepared byinserting the DNA coding for the raffinose synthase into an expressionvector in conformity with the type of cells to be used for expressiontherein. Those usable as the plant expression vector include thosehaving a promoter DNA sequence operative in the plant or a combinationof a plurality of such promoter DNA sequences, and a terminator DNAsequence operative in the plant, and further having a sequence betweenthe both to make it possible to insert a foreign gene.

The promoter includes, for example, promoters which make expression overa whole plant, such as CaMV 35S RNA promoter, CaMV 19S RNA promoter, andnopaline synthase promoter; promoters which make expression in greentissues, such as Rubisco small subunit promoter; and promoters whichmake site-specific expression at portions such as seed, including, forexample, those for genes of napin and phaseolin. The terminatordescribed above includes, for example, nopaline synthase terminator, andRubisco small subunit 3′-side portion.

As for the expression vector for plants, for example, pBI121 andp35S-GFP (produced by CLONTECH) are commercially available, and they maybe used. Alternatively, a vector for expressing virus RNA may be used sothat a gene for an outer coat protein encoded thereby, for example, maybe replaced with the raffinose synthase gene.

In order to achieve transformation, it is advantageous to use methodswhich are usually used for transformation, such as the Agrobacteriummethod, the particle gun method, the electroporation method, and the PEGmethod, in conformity with a host cell to be manipulated. The raffinosesynthase activity can be detected by using the method adopted in thepurification process for the raffinose synthase. Upon the detection, itis desirable to previously remove α-galactosidase, for example, byallowing the sample to pass through an anion exchange column.

The gene coding for the raffinose synthase of cucumber origin includesall of those which provide the raffinose synthase activity uponexpression. Preferably, the gene is exemplified by the gene comprising aDNA coding for the amino acid sequence shown in SEQ ID NO: 5 in SequenceListing, and the gene having the nucleotide sequence shown in SEQ ID NO:4 in Sequence Listing. The gene coding for the raffinose synthase ofsoybean origin includes all of those which provide the raffinosesynthase activity upon expression. Preferably, the gene is exemplifiedby the gene comprising a DNA coding for the amino acid sequence shown inSEQ ID NO: 24 in Sequence Listing, and the gene having the nucleotidesequence shown in SEQ ID NO: 23 in Sequence Listing. It is noted thatthe gene coding for the amino acid sequence shown in SEQ ID NO: 5 or 24in Sequence Listing includes various nucleotide sequences takingdegeneracy of codons into consideration. Namely, the gene coding for theamino acid sequence shown in SEQ ID NO: 5 or 24 in Sequence Listing maybe selected from such various nucleotide sequences, while consideringseveral factors for the gene expression system, such as preferentialcodons depending on, for example, the type of the host cell, andavoidance of higher-order structure to be formed by transcribed RNA. Theselected nucleotide sequence may be a DNA cloned from the nature, or aDNA chemically synthesized in an artificial manner.

<2> DNA Coding for Raffinose Synthase of the Present Invention

The DNA coding for the raffinose synthase can be obtained by preparing acDNA library from poly(A)⁺RNA isolated from a plant such as cucumber,and screening the cDNA library by means of hybridization. A probe to beused for the hybridization can be obtained by performing amplificationby means of PCR (polymerase chain reaction) by using, as primers,oligonucleotides synthesized on the basis of partial amino acidsequences of the raffinose synthase protein.

A method for obtaining the DNA of the present invention from poly(A)⁺RNAoriginating from cucumber will be specifically explained below.

As for the portion for extracting poly(A)⁺RNA, all portions of acucumber plant body may be used provided that the raffinose synthasegene is expressed at that portion. Poly(A)⁺RNA can be obtained, forexample, from leaves, stalks, buds, fruits, and seeds at various growthstages. However, poly(A)⁺RNA is desirably obtained from a material offully expanded leaves after fruiting, especially leaf vein portions.

In order to extract total RNA from the cucumber tissue, any method maybe used without limitation provided that RNA can be efficiently obtainedwith less damage. It is possible to use any known method such as thephenol/SDS method and the guanidine isothiocyanate/cesium chloridemethod. Poly(A)⁺RNA can be isolated from the total RNA thus obtained, byusing an oligo(dT) carrier. It is also preferable to use a kit (forexample, MPG Direct mRNA Purification Kit, produced by CPG, INC.) whichmakes it possible to obtain poly(A)⁺RNA without extracting the totalRNA.

A DNA fragment, which is used as a probe for screening for the cDNAlibrary, can be obtained by performing PCR. Oligonucleotides, which havenucleotide sequences deduced from already known amino acid sequences ofpeptide fragments, for example, nucleotide sequences deduced from aminoacid sequences shown in SEQ ID NOs: 1 to 3, are chemically synthesized.The obtained oligonucleotides are used as primers to perform PCR. Anyportion of the amino acid sequence of the obtained peptide fragment maybe used for the primers. However, it is desirable to select sequenceswhich include less degeneracy of codons and which are assumed to form nocomplicated higher-order structure. Alternatively, it is also preferableto perform RACE (Rapid Amplification of cDNA End, “PCR PROTOCOLS A Guideto Methods and Applications”, ACADEMIC press INC., pp. 28 to 38).

It is desirable to use, as a template for PCR, a cDNA library or singlestrand cDNA. When a heat-resistant DNA polymerase having a reversetranscriptase activity is used for the PCR reaction, it is allowable touse poly(A)⁺RNA, or total RNA in some cases.

In order to prepare the cDNA library, at first single strand cDNAs aresynthesized by using reverse transcriptase while using poly(A)⁺RNA as atemplate and using oligo(dT) primer, random primers or the like. Next,double strand cDNAs are synthesized in accordance with, for example, theGubler and Hoffman method, the Okayama-Berg method (“Molecular Cloning”,2nd edition, Cold Spring Harbor press, 1989). When the raffinosesynthase gene is expressed in a small amount, cDNAs may be amplified bymeans of PCR by using a cDNA library construction kit using PCR (forexample, Capfinder PCR cDNA Library Construction Kit (produced byCLONTECH)). cDNAs thus synthesized can be cloned into a cloning vectorsuch as phage vectors and plasmids, after performing, for example, bluntend formation, addition of a linker, addition of a restriction enzymesite by means of PCR.

A portion characteristic of the raffinose synthase cDNA is selected fromthe DNA fragments obtained by PCR described above, for the probe forhybridization. It is desirable to select a DNA fragment located near tothe 5′-terminal side. The amplified DNA fragment thus selected ispurified from a reaction solution of PCR. In this procedure, theamplified DNA fragment may be purified by subcloning the DNA fragment byusing a plasmid, preparing a large amount of the subcloned plasmid,digesting the prepared plasmid with a restriction enzyme, and excisingthe DNA fragment from a gel after electrophoresis. Alternatively, PCRmay be performed by using the plasmid as a template to amplify and useonly the objective portion. When the amount of the initially amplifiedDNA fragment is sufficiently large, the amplified DNA fragment may bepurified by electrophoresing the DNA fragment without performingsubcloning, excising a gel segment containing a band of the objectiveDNA fragment, and purifying the DNA fragment from the gel segment.

Screening to obtain the objective clone from the cDNA library isperformed by means of hybridization. The DNA fragment obtained inaccordance with the foregoing method can be labeled and used as a probefor the hybridization. Upon labeling, it is possible to use variouslabels such as radioisotope and biotin. However, labeling is desirablyperformed in accordance with the random priming method. Screening may beperformed by using PCR instead of hybridization. Further, screening maybe performed by using hybridization and PCR in combination.

The nucleotide sequence of the DNA coding for the raffinose synthase ofcucumber origin obtained as described above, and the amino acid sequencededuced from the nucleotide sequence are illustratively shown in SEQ IDNO: 4 in Sequence Listing. Only the amino acid sequence is shown in SEQID NO: 5. A transformant AJ13263 of Escherichia coli JM109, whichharbors a plasmid pMossloxCRS containing the DNA fragment including theDNA coding for the raffinose synthase obtained in Example 3 describedlater, has been internationally deposited on the basis of the BudapestTreaty since Nov. 19, 1996 in National Institute of Bioscience and HumanTechnology of Agency of Industrial Science and Technology of Ministry ofInternational Trade and Industry (postal code: 305, 1-3 Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan), and awarded an accession number ofFERM BP-5748.

Furthermore, by using the raffinose synthase gene obtained from oneplant as described above, a raffinose synthase gene can be obtained fromanother plant. As the plant for obtaining the raffinose synthase, any ofplants producing raffinose as described above may be used. For example,soybean, broad bean, rapeseed, sunflower, cotton, sugar beet and thelike may be mentioned. As an example, acquisition of the DNA encodingthe raffinose synthase gene of soybean by using the DNA encoding theraffinose synthase gene of cucumber origin is described.

The raffinose synthase gene of soybean can be obtained by preparing acDNA library from poly(A)⁺RNA derived from soybean, and screening thecDNA library by using a probe selected based on the nucleotide sequenceof the DNA encoding the raffinose synthase gene of cucumber origin.

As a portion from which RNA is extracted, any portion of soybean plantbody can be used provided that the raffinose synthase is expressed.Preferably, a seed, in particular, an immature seed after bloom whichproduces raffinose family oligosaccharides, may be used.

The method for extracting total RNA from the soybean immature seed isnot limited provided that less-damaged. RNA can be efficiently obtained.Any of the methods described above with respect to cucumber may be used.

A probe for hybridization needs to have a nucleotide sequence having ahigh homology with the raffinose synthase gene of soybean origin. Theprobe used for hybridization may be the raffinose synthase gene ofcucumber origin. Preferably, a sequence of a region conserved in theraffinose synthase in the gene may be used as a probe. However, thesequence can not be determined based on only information about theraffinose synthase gene of cucumber origin. To obtain the probe forhybridization having the sequence, the following methods needs to beused. Conveniently, Northern hybridization to soybean RNA is carried outwith fragments obtained by digesting the raffinose synthase gene ofcucumber origin with a suitable restriction enzyme, and a DNA fragmentwhich hybridizes may be used as the probe. Alternatively, the probe maybe obtained by RT-PCR using primers synthesized based on an amino acidsequence of the raffinose synthase of cucumber origin and soybean RNA asa template. Also, the probe may be obtained by RT-PCR usingoligonucleotides synthesized based on Arabidopsis thaliana EST sequenceshaving homology with the DNA encoding the raffinose synthase of cucumberorigin as primers and Alabidopsis thaliana RNA.

Preferably, one having a high homology with the objective gene isobtained as follows. First, EST sequences of Arabidopsis thaliana or thelike which has homology with the raffinose synthase gene of cucumberorigin in GenBank are screened with software such as Genetix Mac or thelike. Regions of a high homology between the obtained sequences and theraffinose synthase gene of cucumber are considered to include a regionconserved among raffinose synthases originating from various species. ADNA fragment of this region can be obtained by, for example,amplification by PCR using single strand DNA prepared from Arabidopsisthaliana RNA as a template and oligonucleotides synthesized based on thesequence of a high homology as primers. The nucleotide sequence of theamplified fragment is analyzed to select one having a sequence of a highhomology with that of cucumber. The obtained DNA fragment is labeled asdescribed above to use as the probe.

For screening of the objective clone from a cDNA library, hybridizationmay be carried out in the same manner as cloning the gene of cucumber.

A nucleotide sequence of a DNA encoding the raffinose synthase ofsoybean origin obtained as described above, and an amino acid sequencededuced from the nucleotide sequence are shown in SEQ ID NO: 23 inSequence Listing. Only the amino acid sequence is shown in SEQ ID NO:24. Homology of the raffinose synthase of soybean origin with theraffinose synthase of cucumber origin is 38% in the amino acid sequenceand 50% in the nucleotide sequence by maximum matching which allowsgaps. The transformant, designated as AJ13379, of Escherichia coliJM109, which harbors the plasmid pMOSSloxSRS containing a DNA fragmentcontaining DNA coding for the raffinose synthase obtained in Example 4as described below, has been internationally deposited on the basis ofthe Budapest Treaty since Oct. 20, 1997 in National Institute ofBioscience and Human Technology of Agency of Industrial Science andTechnology of Ministry of International Trade and Industry (postal code:305, 1-3 Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), and awardedan accession number of FERM BP-6149.

By using information about the raffinose synthase of cucumber origin andthe raffinose synthase of soybean origin, a raffinose synthase gene maybe obtained from another plant. Single strand DNA having a nucleotidesequence deduced from the amino acid sequence conserved between the bothproteins, for example, SEQ ID NO: 28 (amino acid numbers 199 to 208 ofSEQ ID NO: 24), 29 (amino acid numbers 302 to 314 of SEQ ID NO: 24), 30(amino acid numbers 513 to 527 of SEQ ID NO: 24) in Sequence Listing, orsingle strand DNA having a nucleotide sequence complementary to thededuced nucleotide sequence may be synthesized and RT-PCR may be carriedout by using the single strand DNAs as primers. Any portion of thesequence may be used for the primer. Preferably, a sequence in whichdegeneracy of codon is small and complicated high-order structure is notconsidered to be formed. PCR is carried out by using cDNA synthesizedfrom total RNA or poly(A)⁺RNA in some cases of a plant from which thegene is to be obtained, as a template. The obtained DNA fragment iscloned into a suitable vector to analyze nucleotide sequence, therebyconfirming that the nucleotide sequence has homology with the raffinosesynthase gene originating from cucumber or soybean or an amino acidsequence translated therefrom has homology with the amino acid sequenceof the raffinose synthase originating from cucumber or soybean. Thusobtained DNA fragment can be used for screening of a cDNA library.Alternatively, RACE may be carried out using single strand cDNAsynthesized from total RNA or poly(A)⁺RNA in some cases of a plant fromwhich the gene is to be obtained, as a template.

The DNA of the present invention may code for a raf finose synthaseprotein including substitution, deletion, insertion, addition, orinversion of one or several residues of amino acids at one or severalpositions, provided that the activity of raffinose synthase encodedthereby, i.e., the activity to produce raffinose from sucrose andgalactinol is not deteriorated. In this context, the number of “severalresidues” differs depending on the position and the type of the aminoacid residues in the three-dimensional dimensional structure of theprotein, originally because of the following reason. Namely, highsimilarity is found between some amino acids and other amino acids, forexample, between isoleucine and valine, and such a difference in aminoacid does not greatly affect the three-dimensional structure of theprotein. Therefore, the DNA of the present invention may code for thosehaving homology of not less than 35 to 40% with respect to the entire784 amino acid residues for constituting the raffinose synthase ofcucumber origin, provided that they have the raffinose synthaseactivity. Preferably, they have homology of 65% in a region between510th amino acid and 610th amino acid. Also, the DNA of the presentinvention may code for those having homology of not less than 35 to 40%with respect to the entire 750 amino acid residues for constituting theraffinose synthase of soybean origin, provided that they have theraffinose synthase activity. Preferably, they have homology of 65% in aregion between 478th amino acid and 577th amino acid. Specifically, thenumber of “several residues” is 2 to 40, preferably 2 to 20, and morepreferably 2 to 10. The homology is a value determined by the maximummatching which allows gaps.

The present invention includes genes in which homology of not less thanabout 50% is given for the entire length of the gene, and homology ofnot less than 65% is given over a region comprising about 300 nucleotideresidues. Nucleotide sequence information on such genes can be obtainedby searching genes having homology to the raffinose synthase gene ofcucumber origin, by using a database such as GenBank. For example,GENETIX-MAC (software for processing genetic information, produced bySoftware Development), which adopts the Lipman-Person method, may beused as a program for homology analysis. Alternatively, those open tothe public on the Internet may be used for this purpose. Somenucleotides sequences obtained by the method as described above containthe entire length of the gene, and other nucleotide sequences do notcontain the entire length of the gene. When the entire length of thegene is not contained, the entire length gene can be easily obtained byusing RNA extracted from an objective plant tissue as a template, andusing primers corresponding to portions having high homology to theraffinose synthase gene of cucumber origin, in accordance with the5′-RACE method and the 3′-RACE method. The obtained entire length genemay be incorporated into an appropriate expression vector provided asthose included in a kit such as Soluble Protein Expression System(produced by INVITROGEN), Tight Control Expression System (produced byINVITROGEN), and QIAexpress System (produced by QIAGEN) as describedabove, so that the gene may be expressed, and then the raffinosesynthase activity may be measured in accordance with the methoddescribed above to select a clone having the activity. The methods forgene expression are detailed in Plant Molecular Biology, A LaboratoryManual (Melody S. Clark (Ed.), Springer) and the like.

A DNA, which codes for substantially the same protein as the raffinosesynthase, can be obtained by modifying the nucleotide sequence inaccordance with, for example, the site-directed mutagenesis method sothat amino acids located at specified positions are subjected tosubstitution, deletion, insertion, or addition. The modified DNA asdescribed above may be also obtained in accordance with theconventionally known mutation treatment. The mutation treatment includesa method in which the DNA coding for the raffinose synthase is treatedwith hydroxylamine or the like in vitro, and a method in which abacterium belonging to the genus Escherichia harboring the DNA codingfor the raffinose synthase is treated with ultraviolet irradiation or amutating agent usually used for artificial mutation, such as nitrousacid and N-methyl-N′-nitro-N-nitrosoguanidine (NTG).

The substitution, deletion, insertion, addition, or inversion of thenucleotide includes mutation which naturally occurs, for example, basedon the difference between individuals of a cucumber or soybean plant,the difference between varieties, the formation of multiple copies ofthe gene, the difference between respective organs, and the differencebetween respective tissues.

DNA having mutation as described above is expressed in an appropriatecell to investigate the raffinose synthase activity of an expressedproduct. Thus it is possible to obtain a DNA which codes forsubstantially the same protein as the raffinose synthase. Further, theDNA coding for substantially the same protein as the raffinose synthaseprotein can be obtained by isolating a DNA which hybridizes understringent conditions with a DNA having a nucleotide sequence comprisingnucleotide residues of nucleotide numbers 56 to 2407 in the nucleotidesequence shown in SEQ ID NO: 4 or a nucleotide sequence comprisingnucleotide residues of nucleotide numbers 156 to 2405 in the nucleotidesequence shown in SEQ ID NO: 23 in Sequence Listing, for example, andwhich codes for the protein having the raffinose synthase activity, fromDNAs coding for raffinose synthases having mutation or from cellsharboring the DNAs. The phrase “stringent conditions” referred to hereinindicates a condition in which the specific hybrid is formed, andnonspecific hybrid is not formed. It is difficult to definitely expressthis condition by using numerical values. However, for example, thiscondition includes a condition in which DNAs having high homology, forexample, DNAs having homology of not less than 50% hybridize with eachother, while DNAs having homology lower than the above do not hybridizewith each other, or a condition in which hybridization is achieved at asalt concentration corresponding to a washing condition for ordinarySouthern hybridization, i.e., 1×SSC, 0.1% SDS, and preferably 0.1×SSC,0.1% SDS, at 60° C. Genes, which hybridize under such a condition, mayinclude those which contain a stop codon generated at an intermediateposition, and those which have lost the activity due to mutation at theactive center. However, those having such mutation can be easilyeliminated by ligating the gene with a commercially available activityexpression vector to measure the raffinose synthase activity inaccordance with the method described above.

When the DNA of the present invention is used to express an antisenseRNA for the raffinose synthase, it is unnecessary for the DNA to codefor any active raffinose synthase. Further, the function of anyendogenous gene having homology can be restrained by using a sense RNA.In such a case, it is also unnecessary for the DNA to code for anyactive raffinose synthase. Further, it is unnecessary for the DNA tocontain the entire length. Preferably, it is sufficient for the DNA tohave about 500 base pairs of a translating region having 60% ofhomology. An example of the DNA is a DNA of the following (e) or (f):

(e) a DNA which hybridizes under stringent conditions with a nucleotidesequence comprising at least nucleotide residues of nucleotide numbers56 to 2407 in a nucleotide sequence shown in SEQ ID NO: 4 in SequenceListing, or a complementary nucleotide sequence thereof; or

(f) a DNA which hybridizes under stringent conditions with a nucleotidesequence comprising at least nucleotide residues of nucleotide numbers156 to 2405 in a nucleotide sequence shown in SEQ ID NO: 23 in SequenceListing, or a complementary nucleotide sequence thereof.

The method has been explained above, in accordance with which thepresent inventors have succeeded in cloning the objective cDNA of theraffinose synthase originating from cucumber or soybean. However, otherthan the foregoing, the following methods may be available.

(1) The raffinose synthase originating from cucumber or soybean isisolated and purified, and an entire nucleotide sequence is chemicallysynthesized on the basis of a determined amino acid sequence or theamino acid sequence shown in SEQ ID NO: 5 or 24.

(2) Chromosomal DNA is prepared from a cucumber or soybean plant body,and a chromosomal DNA library is prepared by using a plasmid vector orthe like. The raffinose synthase gene is obtained from the library bymeans of hybridization or PCR. It is assumed that the raffinose synthasegene originating from chromosome contains intron in its coding region.However, DNA divided into several parts by such intron is included inthe DNA of the present invention provided that it codes for theraffinose synthase.

(3) Poly(A)⁺RNA is fractionated into fractions in accordance with themolecular weight or the like. The fractions are subjected to an in vitrotranslation system using wheat germ or rabbit reticulocyte to determinea fraction containing mRNA coding for a polypeptide having the raffinosesynthase activity. An objective cDNA fragment is prepared and obtainedfrom the fraction.

(4) An anti-cucumber raffinose synthase antibody or an anti-soybeanraffinose synthase antibody is prepared. Elements of a cDNA library areincorporated into a protein expression vector, and an appropriate hostis transfected therewith to express proteins encoded by cDNAs. Anobjective cDNA may be screened by using the foregoing antibody.

(5) Appropriate primers are synthesized on the basis of amino acidsequences of peptide fragments, and a sequence containing the terminalis amplified by means of the RACE method, followed by cloning thereof.

For expression of the raffinose synthase gene, a DNA of a regionencoding the enzyme may be introduced to various expression vectors toexpress the gene. Specifically, it is described in Plant MolecularBiology-A Laboratory Manual (M. S. Clark (eds.), Springer) and the like.As the vector, commercially available expression vectors may be used.Confirmation of the expression can be carried out by measuring anactivity according to the method described in the present specification.

As an example, a method for expression of the raffinose synthaseactivity by the raffinose synthase gene originating from soybean isdescribed. An NdeI restriction enzyme site and a BamHI site are added toimmediately upstream portion including ATG of 156th nucleotide anddownstream portion of 2405th nucleotide, respectively, by PCR usingprimers designed to have the respective restriction enzyme sites. Then,the raffinose synthase gene purified by the phenol-chloroform method andpET3a are each digested with NdeI and BamHI. The digested DNAs are eachpurified by agarose gel electrophoresis. Since a BamHI site is presentin the raffinose synthase gene originating from soybean, mutation ispreviously made by PCR or the like, or a fragment having an objectivesize is selected by agarose gel electrophoresis. The purified raffinosesynthase gene fragment is ligated to the vector, and the ligation isconfirmed by agarose gel electrophoresis. Also, sequencing is carriedout to confirm that the raffinose synthase gene starts from ATG codon.E. coli BL21 (DE3) pLysE is transformed with the vector, andtransformants are selected with LB medium containing chloramphenicol andampicillin. The insert fragment in the transformant is confirmed by PCRor the like, the objective transformant is cultured to obtain cells. Thecells are incubated with a gel-loading buffer containing SDS at 100° C.for 3 minutes. Then, SDS polyacrylamide gel electrophoresis is carriedout to confirm a protein band of an objective size. The selected strainis cultured, and protein is extracted by disrupting cells withsonication or the like. The raffinose synthase activity of the extractedprotein solution may be determined by the method described in thepresent specification.

<3> Method for Producing Raffinose of the Present Invention

In the method for producing raffinose of the present invention, theraffinose synthase is allowed to act on sucrose and galactinol toproduce raffinose. When the raffinose synthase is allowed to act onsucrose and galactinol, the galactose residue constituting galactinol istransferred to sucrose, and thus raffinose is produced. During thisprocess, myo-inositol constituting galactinol is liberated.

The raffinose synthase, which is used to produce raffinose, may be anenzyme extracted from a plant body, or an enzyme produced by means ofthe genetic recombination technique based on the use of the DNA of thepresent invention.

In order to allow the raffinose synthase to act on sucrose andgalactinol, the following procedure may be available. Namely, theraffinose synthase or cells having an ability to produce the raffinosesynthase are immobilized to a carrier such as alginic acid gel andpolyacrylamide gel to prepare immobilized enzyme or immobilized cells.The immobilized enzyme or the immobilized cells are charged to a column,and a solution containing sucrose and galactinol is allowed to passthrough the column. As for the carrier and the method for immobilizingthe raffinose synthase or the cells to the carrier, it is possible toadopt materials and methods which are used for ordinary bioreactors.

The raffinose synthesis reaction is performed, for example, by addingthe raffinose synthase to a solution such as an aqueous solution or abuffer containing sucrose and galactinol. It is preferable that pH ofthe solution is adjusted to be within a range of about 6 to 8,especially at about pH 7. The reaction temperature is within a range ofabout 28 to 42° C., preferably 35 to 40° C., especially about 38° C. Theraffinose synthase of the present invention is stable within a range ofpH 5 to 8, especially in the vicinity of pH 6. The enzyme of the presentinvention is stable within a temperature range of not more than about40° C.

The enzyme activity of the raffinose synthase of the present inventionis inhibited by iodoacetamide, N-ethylmaleimide, MnCl₂, ZnCl₂, andNiCl₂. Therefore, it is desirable that these substances are notcontained in the reaction solution.

Preferably, galactinol and sucrose are added to the reaction solution ata concentration of not less than 5 mM of galactinol and a concentrationof not less than 1.5 mM of sucrose. The raffinose synthase may be addedto the reaction solution in an amount depending on the amounts of thesubstrates.

Raffinose is separated from unreacted sucrose and galactinol and frommyo-inositol produced by the enzyme reaction, contained in the reactionsolution, in accordance with a method including, for example, gelfiltration chromatography.

<4> Chimeric Gene and Transgenic Plant of the Present Invention

The chimeric gene of the present invention includes the raffinosesynthase gene or a part thereof and the transcription regulatory regionexpressible in plant cells. The raffinose synthase gene is exemplifiedby the DNA coding for the raffinose synthase of the present inventiondescribed in the foregoing item <2>. When the chimeric gene of thepresent invention is used as an antisense gene, a non-coding region ofthe raffinose synthase gene or a part thereof can be used in some cases,besides the DNA coding for the raffinose synthase. The non-coding regionincludes, for example, sequences indicated by nucleotide numbers 1 to 55(5′-non-coding region) and nucleotide numbers 2407 to 2517(3′-non-coding region) in SEQ ID NO: 4 as well as nucleotide numbers 1to 155 and nucleotide numbers 2406 to 2765 in SEQ ID NO: 23 in SequenceListing.

When the transcription regulatory region is linked to the DNA coding forthe raffinose synthase in the chimeric gene of the present invention sothat mRNA (sense RNA) homologous to the coding strand of the DNA isexpressed, plant cells to which the chimeric gene is introduced expressthe raffinose synthase, and the content of the raffinose familyoligosaccharides is increased. On the other hand, when thetranscriptional regulatory region is linked to the DNA so that RNA(antisense RNA) having a sequence complementary to the coding strand ofthe DNA is expressed, and when the transcription regulatory region islinked to the DNA so that a partial fragment of the raffinose synthasegene, preferably sense RNA for a portion of not less than about 200 basepairs in the upstream coding region is expressed, then the expression ofendogenous raffinose synthase is restrained in plant cells to which thechimeric gene is introduced, and the raffinose family oligosaccharidesare decreased.

The content of the raffinose family oligosaccharides in a plant can bechanged by transforming the plant with the chimeric gene of the presentinvention, and allowing the gene to be expressed in cells of the plant.

Plants to which the present invention is applicable include, forexample, oil crops such as soybean, rapeseed, cotton; sugar crops suchas sugar beet and sugar cane; and model plants represented byArabidopsis thaliana.

The transcription regulatory region expressible in plant cells includes,for example, promoters which make expression over a whole plant, such asCaMV 35S RNA promoter, CaMV 19S RNA promoter, and nopaline synthasepromoter; promoters which make expression in green tissues, such asRubisco small subunit promoter; and promoter regions which makesite-specific expression at portions such as seed, including, forexample, those for genes of napin and phaseolin as described above. The3′-terminal of the chimeric gene may be connected with the terminatorsuch as nopaline synthase terminator, and Rubisco small subunit 3′-endportion.

The plant may be transformed with the chimeric gene in accordance withusually used methods such as the Agrobacterium method, the particle gunmethod, the electroporation method, and the PEG method, depending on thehost cell to be manipulated.

The transformation method for introducing the chimeric gene into theplant includes, for example, the Agrobacterium method, the particle gunmethod, the electroporation method, and the PEG method.

The Agrobacterium method is specifically exemplified by a method using abinary vector. Namely, a plant is transfected with a vector comprisingT-DNA originating from Ti plasmid, a replication origin which isfunctional in microorganisms such as Escherichia coli, and a marker genefor selecting plant cells or microbial cells harboring the vector. Seedsare collected from the plant, and they are allowed to grow. Plants towhich the vector is introduced are selected by using an index ofexpression of the marker gene. Obtained plants are measured for theraffinose synthase activity, or strains exhibiting change in content ofthe raffinose family oligosaccharides are selected from the obtainedplants. Thus it is possible to obtain an objective transformed plant.

A method for introducing the chimeric gene into soybean will bedescribed below. In order to perform transformation for soybean, it ispossible to use any of the particle gun method (Pro. Natl. Acad. Sci.USA, 86, 145 (1989); TIBTECH, 8, 145 (1990); Bio/Technology, 6, 923(1988); Plant Physiol, 87, 671 (1988); Develop. Genetics, 11, 289(1990); and Plant cell Tissue & Organ Culture, 33, 227 (1993)), theAgrobacterium method (Plant Physiol., 91, 1212 (1989); WO 94/02620;Plant Mol. Biol., 9, 135 (1987); and Bio/Technology, 6, 915 (1988)), andthe electroporation method (Plant Physiol, 99, 81 (1992); Plant Physiol,84, 856 (1989); and Plant Cell Reports, 10, 97 (1991)).

In the particle gun method, it is preferable to use an embryogenictissue or a hypocotyl of an immature seed about 30 to 40 days afterdehiscence of anthesis. About 1 g of the embryogenic tissue is spreadover a petri dish, and, for example, gold particles or tungstenparticles coated with the objective chimeric gene may be shot thereinto.The tissue is transferred after 1 to 2 hours to a liquid medium toperform cultivation. After 2 weeks, the tissue is transferred to amedium containing an antibiotic for transformant selection, followed bycultivation. After 6 weeks, a green adventitious embryo which isresistant to the antibiotic is obtained. The adventitious embryo isfurther transferred to a fresh medium and cultured so that a plant bodyis reproduced. Alternatively, when the hypocotyl is used, the hypocotylis excised under a sterilized condition, and it is treated in accordancewith the particle gun method, followed by cultivation in MS medium(Murashige and Skoog, Physiologia Plantrum, 15, 473-497 (1962))containing cytokinin at a high concentration. The hypocotyl is culturedin the darkness for 2 weeks, and then it is cultured at room temperaturewith light irradiation for 12 to 16 hours in MS medium having a loweredcytokinin content. During this process, it is preferable to add, to themedium, the antibiotic having been used as the selection marker. When amultiple bud body is formed from the transplanted tissue, it istransferred to a medium supplemented with no hormone so that rooting iscaused. An obtained seedling body is transferred to a greenhouse andcultivated.

In the case of the method using Agrobacterium, it is desirable to usecotyldonary nod as a plant tissue. Commercially available LBA4404, C58,and Z707 can be used as Agrobacterium. It is desirable to use Z707. Forexample, a plasmid obtained by inserting the objective gene into pMON530(produced by Monsanto Co.) can be used as the vector. The plasmid isintroduced into Agrobacterium tumefaciens Z707 (Hepburn et al., J. Gen.Microbiol., 131, 2961 (1985)) in accordance with, for example, thedirect freeze thaw method (An et al., “Plant Mol. Biol. Mannual”, A3:1-19, 1988). The Agrobacterium transformed with the chimeric gene iscultivated overnight. Proliferated cells are collected by centrifugationat 5000 rpm for 5 minutes, and they are suspended in B5 suspensionmedium. Soybean seeds are sterilized, and they are cultivated for 3 dayson B5 medium having a 1/10 concentration so that they germinate.Cotyledons are excised, and they are cultivated for 2 hours with thesuspension of Agrobacterium. The cotyledons are transferred to B5 medium(containing Gamborg B5 salt (Exp. Cell. Res., 50, 151 (1968)), GamborgB5 vitamin, 3% sucrose, 5 μM benzylaminopurine, 10 μM IBA, and 100 μMacetosyringon), and they are cultivated for 3 days under a condition at25° C. with light irradiation (60 μEm⁻²S⁻¹) for 23 hours. Subsequently,in order to remove Agrobacterium, the cotyledons are cultivated in B5medium (5 μM benzylaminopurine, 100 mg/L carbenicillin, 100 mg/Lvancomycin, and 500 mg/L cefotaxime) at 25° C. for 4 days whileexchanging the medium every day. After that, the cotyledons arecultivated in B5 medium (200 mg/L kanamycin). Multishoots are formedwithin 1 or 2 months. They are cultivated on B5 medium (0.58 mg/Lgibberellin and 50 mg/L kanamycin) to elongate the shoots. Subsequently,the shoots are transferred to B5 medium (10 μM IBA) to cause rooting.Rooted seedlings are acclimatized, and they are cultivated in agreenhouse. Thus transformants can be obtained.

A transformant plant, in which the raffinose synthase gene isintroduced, can be easily confirmed by extracting DNA from thetransformant, and performing Southern hybridization by using theraffinose synthase gene as a probe.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a relationship between the reaction time and the amount ofraffinose produced by the raffinose synthesis reaction.

FIG. 2 shows a diagram illustrating a result of SDS-polyacrylamide gelelectrophoresis for the raffinose synthase. M indicates molecular weightmarkers, and S indicates a sample containing the raffinose synthase.Numerals indicate molecular weights (kDa).

FIG. 3 shows an influence of the reaction temperature on the raffinosesynthase activity.

FIG. 4 shows an influence of the reaction pH on the raffinose synthaseactivity.

FIG. 5 shows an influence of myo-inositol on the raffinose synthaseactivity.

FIG. 6 shows a stable pH range of the raffinose synthase.

FIG. 7 shows relationships between synthetic primers and amino acidsequences of peptides. R represents A or G, Y represents C or T, Mrepresents A or C, K represents G or T, D represents G, A, or T, Hrepresents A, T, or C, B represents G, T, or C, N represents G, A, T, orC, and I represents inosine.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be more specifically explained below withreference to Examples.

At first, the method for measuring the raffinose synthase activity, usedto confirm active fractions during respective purification steps andinvestigate characteristics of the enzyme in the following Examples,will be explained.

<Method for Measuring the Raffinose Synthase Activity>

The activity of the raffinose synthase was measured by quantitativelydetermining raffinose produced by the raffinose synthesis reaction byusing HPLC (high-performance liquid chromatography). HPLC was performedby using Sugar Analysis System DX500 (CarboPac PA1column, pulsedamperometry detector (produced by DIONEX)).

For the raffinose synthesis reaction, to a reaction solution prepared tohave a composition having the following final concentrations, 10 to 50μl of a raffinose synthase solution was added to give a volume of 100 μlfollowed by performing the reaction at 32° C. for 60 minutes.

[Composition of Reaction Solution (Final Concentration)]

2.5 mM sucrose   5 mM galactinol   5 mM DTT  20 mM Tris-HCl buffer (pH7.0)

After performing the reaction as described above, the reaction wasstopped by adding to the reaction solution, ethanol in a volume fourtimes the volume of the reaction solution and heating the solution at95° C. for 30 seconds. The obtained solution was centrifuged to obtain asupernatant and the supernatant was then dried up under a reducedpressure. After that, an obtained residue was dissolved in distilledwater. Raffinose in the reaction product was quantitatively determinedby using the sugar analysis system to estimate the raffinose synthaseactivity.

EXAMPLE 1 Purification of Raffinose Synthase from Cucumber

<1> Extraction of Raffinose Synthase from Cucumber

Vein tissues were collected from true leaves of cucumber (cv.: SUYOU)obtained 6 to 10 weeks after planting. The leaf vein tissues were frozenwith liquid nitrogen, and they were stored at −80° C. The frozen leafvein tissues were ground by a mortar with liquid nitrogen, and Buffer 1(40 mM Tris-HCl buffer (pH 7.0), 5 mM DTT, 1 mM PMSF(phenylmethanesulfonyl fluoride), 1% polyclarl AT (produced by Serva))was added thereto to extract proteins. An obtained extract solution wasfiltrated with a filter such as gauze or Miracloth (produced byCalbiochem-Novobiochem). An obtained filtrate was centrifuged at 4° C.at about 30,000×g for 60 minutes. A supernatant obtained by thecentrifugation was used as a crude extract solution.

<2> Anion Exchange Chromatography (1)

The crude extract solution (about 560 ml) obtained as described abovewas applied to a column system comprising five connected columns forstrongly basic anion exchange chromatography (HiTrap Q, produced byPharmacia, 1.6 cm×2.5 cm) equilibrated with Buffer 2 (20 mM Tris-HClbuffer (pH 7.0), 5 mM DTT) to adsorb the raffinose synthase activity tothe columns. Subsequently, the columns were washed with Buffer 3 (20 mMTris-HCl buffer (pH 7.0), 0.2 M NaCl, 5 mM DTT) in a volume five timesof the columns so that non-adsorbed proteins were washed out. Afterthat, the raffinose synthase activity was eluted from the columns with50 ml of Buffer 4 (20 mM Tris-HCl buffer (pH 7.0), 0.3 M NaCl, 5 mMDTT).

<3> Anion Exchange Chromatography (2)

The eluted solution (about 75 ml) was placed in a dialysis tube(Pormembranes MWC O: 10,000, produced by Spectra), and it was dialyzedagainst 10 L of Buffer 5 (20 mM Tris-HCl buffer (pH 7.0), 0.05 M NaCl, 5mM DTT) at 4° C. overnight. The dialyzed sample was applied to a columnfor weakly basic anion exchange chromatography (DEAE-TOYOPEARL, producedby Tosoh Corp., 2.2×20 cm) equilibrated with Buffer 5 to adsorb theraffinose synthase activity to the column. Subsequently, the column waswashed with Buffer 5 in a volume five times the volume of the column towash out non-adsorbed proteins. After that, a linear concentrationgradient of 0.05 M to 0.35 M NaCl in a volume twenty times the volume ofthe column was applied to elute the enzyme activity so thatfractionation was performed.

<4> Gel Filtration Chromatography

The eluted solution obtained as described above (about 160 ml) wasconcentrated into 6.5 ml by using a concentrator (Centriprep 10,produced by Amicon). Aliquots (each 3 ml) of the concentrated solutionwere applied to a column for gel filtration chromatography (Superdex 200pg, produced by Pharmacia, 2.6 cm×60 cm). Equilibration for the columnand elution from the column were performed by using Buffer 6 (20 mMTris-HCl buffer (pH 7.0), 0.1 M NaCl, 5 mM DTT, 0.02% Tween 20).Fractions having the raffinose synthase activity were collected fromfractionated fractions.

<5> Hydroxyapatite Chromatography

A collected fraction (about 25 ml) having the raffinose synthaseactivity fractionated by the gel filtration was concentrated by usingCentriprep 10, and the buffer was exchanged with Buffer 7 (0.01 M sodiumphosphate buffer (pH 7.0), 5 mM DTT, 0.02% Tween 20). An obtainedconcentrate solution (about 1.2 ml) was applied to a hydroxyapatitecolumn (Bio-Scale CHT-1, produced by Bio Rad, 0.7×5.2) previouslyequilibrated with the same buffer to adsorb the raffinose synthaseactivity. The column was washed with the same buffer in a volume (10 ml)five times the volume of the column. After that, a linear concentrationgradient of 0.01 M to 0.3 M phosphate in a volume twenty times thevolume of the column was applied to elute the enzyme activity so thatfractionation was performed.

<6> Hydroxyapatite Rechromatography

An active fraction obtained in accordance with the hydroxyapatitechromatography as described above was subjected to rechromatography inthe same manner as described above to obtain a purified raffinosesynthase fraction (about 2 ml).

The amount of protein contained in the active fraction was about 200 μg.The total activity was 5700 nmol/hour, and the specific activity perprotein was about 28 μmol/hour/mg. The active fraction contained only aprotein which exhibited a single band corresponding to a molecularweight of 90 kDa to 100 kDa on electrophoresis as described later. Thespecific activity of the obtained purified enzyme sample was about 2000times that of the crude extract solution. The recovery was 12% withrespect to the amount of the enzyme obtained after the strongly basicanion exchange chromatography using HiTrap Q. Results of thepurification are summarized in Table 1.

TABLE 1 Total Total Specific protein activity activity mg nmol/hnmol/h/mg Yield % Crude extract 1915 20700 11 — HiTrap Q 1092 48800 45100 DEAE-TOYOPEARL 540 33000 61 68 Superdex 200 pg 1.79 26500 14800 54Apatite (1)* 0.51 12600 24700 26 Apatite (2)* 0.20 5700 28500 12 Apatite(1)*: Hydroxyapatite chromatography (1) Apatite (2)*: Hydroxyapatitechromatography (2)

EXAMPLE 2 Investigation on Characteristics of Raffinose Synthase

Characteristics of the purified raffinose synthase obtained in Example 1were investigated.

<1> Molecular Weight Measurement

(1) Gel Filtration Chromatography

An aliquot (10 μl) of the purified raffinose synthase was dispensed.This sample and a molecular weight marker (Molecular Weight Marker Kitfor Gel Filtration, produced by Pharmacia) were applied to a gelfiltration chromatography column (Superdex 200 pg, produced byPharmacia). Equilibration of the column and elution from the column wereperformed by using Buffer 6 (20 mM Tris-HCl buffer (pH 7.0), 0.1 M NaCl,5 mM DTT, 0.02% Tween 20). As a result, the molecular weight of theraffinose synthase was estimated to be about 75 kDa to 95 kDa.

(2) Polyacrylamide Gel Electrophoresis (Native PAGE)

An aliquot (10 μl) of the purified raffinose synthase was dispensed, andthe same volume of a sample buffer (0.0625 M Tris-HCl (pH 6.8), 15%glycerol, 0.001% BPB) was added thereto to prepare an electrophoresissample. The sample (10 μl) was applied to 10% polyacrylamide gel(produced by Daiichi Chemical, Multigel 10), and electrophoresed at 40mA for about 60 minutes with 0.025 M Tris—0.192 M glycine buffer (pH8.4). After the electrophoresis, the gel was stained with Silver StainKit (produced by nacalai tesque). As a result, the molecular weight wasestimated to be about 90 kDa to 100 kDa.

(3) SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE)

An aliquot (10 μl) of the purified raffinose synthase was dispensed, andthe same volume of a sample buffer (0.0625 M Tris-HCl (pH 6.8), 2% SDS,10% glycerol, 5% mercaptoethanol, 0.001% BPB) was added thereto,followed by heating in a boiling water bath for 1 minute to prepare anelectrophoresis sample. The sample (10 μl) was applied to 10 to 20%gradient polyacrylamide gel (produced by Daiichi Chemical), andelectrophoresed at 40 mA for about 70 minutes with 0.025 M Tris—0.192 Mglycine buffer (pH 8.4) containing 0.1% SDS. After the electrophoresis,the gel was stained with Silver Stain Kit (produced by nacalai tesque).A result is shown in FIG. 2. As a result, the molecular weight wasestimated to be about 90 kDa to 100 kDa.

<2> Optimum Reaction Temperature

The raffinose synthase activity was measured under various temperatureconditions (28° C., 32° C., 36° C., 40° C., 44° C., 48° C., and 52° C.)in accordance with the method for measuring the raffinose synthaseactivity described above. The enzyme solution was added to each of thereaction solutions in an amount of 2 μl. FIG. 3 shows relativeactivities at the respective temperatures assuming that the enzymeactivity at 32° C. was 100. As a result, the raffinose synthaseexhibited the activity in a range of about 25 to 42° C., and the optimumreaction temperature was about 35 to 40° C.

<3> Optimum Reaction pH

The raffinose synthase activity was measured under various pH conditions(pH 4 to 11) in accordance with the method for measuring the raffinosesynthase activity described above. The reactions were performed by using50 mM citrate buffer (pH 4 to 6), 50 mM potassium phosphate buffer (pH5.5 to 7.5), 50 mM Bis-Tris buffer (pH 6 to 7), 20 mM Tris-HCl buffer(pH 7 to 8.5), and 50 mM glycine-NaOH buffer (pH 9 to 11). The enzymesolution was added to the respective reaction solutions in an amount of2 μl. A result is shown in FIG. 4.

As a result, the raffinose synthase exhibited the activity in a range ofpH 5 to 10, and the optimum reaction pH was about 6 to 8, provided thatthe activity varied depending on the type of the buffer used for themeasurement.

<4> Investigation on Inhibitors and Metal Ions

Various enzyme inhibitors or metal ions were added to the reactionsolution of the purified raffinose synthase to give a finalconcentration of 1 mM respectively, and the raffinose synthase activitywas measured in the same manner as described above. Table 2 showsremaining activities with respect to the enzyme activity obtained whenneither inhibitor nor metal ion was added. Iodoacetamide andN-ethylmaleimide remarkably inhibited the enzyme activity. Theinhibiting effect was scarcely observed for CaCl₂, CuCl₂, and MgCl₂.However, MnCl₂, ZnCl₂, and NiCl₂ exhibited the inhibiting effect.

TABLE 2 Inhibitor or metal ion Remaining activity (%) No addition 100Iodoacetoamide 0 N-ethylmaleimide 40 CaCl₂ 115 CuCl₂ 101 MgCl₂ 96 MnCl₂32 ZnCl₂ 42 NiCl₂ 68<5> Inhibition by Myo-inositol

Investigation was made for inhibition by myo-inositol as the reactionproduct of the raffinose synthesis reaction. To the reaction solution,myo-inositol was added at various concentrations, and the raffinosesynthase activity was measured. A result is shown in FIG. 5. The enzymeactivity was inhibited as the concentration of added myo-inositol wasincreased.

<6> Stable pH

The raffinose synthase fraction obtained by the anion exchangechromatography (2) described above was incubated for 4 hours at 4° C. in50 mM Bis-Tris-HCl buffer (pH 5 to 8.0, containing 0.5 mM DTT) or 20 mMTris-HCl buffer (pH 7 to 8.0, containing 0.5 mM DTT), and then theraffinose synthase activity was measured. FIG. 6 shows the enzymeactivity versus pH of the buffer used for the incubation. The raffinosesynthase activity was confirmed after the incubation under any of theincubation conditions. Especially, the enzyme was stable in a range ofpH 5 to 7.5.

<7> Stable Temperature

The raffinose synthase fraction obtained by the anion exchangechromatography (2) described above was incubated in 20 mM Tris-HClbuffer (pH 7, containing 0.5 mM DTT) for 60 minutes at 28° C., 32° C.,37° C., or 40° C., and then the raffinose synthase activity wasmeasured. As a result, the enzyme of the present invention exhibited, inthe range of 28° C. to 40° C., activities of 80% to 100% of thatobtained by a control for which the incubation treatment was notperformed for comparison, and therefore was stable in the range.

<8> Analysis of Amino Acid Sequence

The cysteine residue of the purified raffinose synthase was subjected toreducing pyridylethylation, and the reaction mixture was desalted. Anobtained sample was digested at 37° C. for 12 hours withlysylendopeptidase (Achromobacter protease 1, produced by Wako PureChemical Industries) to form peptide fragments. An obtained peptidemixture was applied to reverse phase HPLC (column: Waters μBondasphere(φ2.1×150 mm, C₁₈, 300 Å, produced by Waters (Millipore))) to separateand obtain the respective peptide fragments. 0.1% TFA (trifluoroaceticacid) was used as a solvent, and elution was performed with aconcentration gradient of acetonitrile. Amino acid sequences of threefragments selected from the obtained peptide fragments were determinedby using a protein sequencer. The determined amino acid sequences of therespective peptides are shown in SEQ ID NOs: 1 to 3 in Sequence Listing.These peptides will be thereafter referred to as Peptides 1, 2, and 3respectively in this order.

EXAMPLE 3 Preparation of DNA Coding for Raffinose Synthase Originatingfrom Cucumber

<1> Isolation of Partial Fragment of cDNA of Raffinose Synthase by Meansof PCR Method

Major veins (22 g) of cucumber were ground by a mortar with liquidnitrogen. The ground material was added to a mixture of an extractionbuffer (100 mM lithium chloride, 100 mM Tris-HCl (pH 8.0), 10 mM EDTA,and 1% SDS) and an equal amount of phenol previously heated to 80° C.,followed by agitation. After that, a mixture of phenol and an equalamount of chloroform:isoamyl alcohol (24:1) was added thereto, followedby agitation again. An obtained mixture solution was centrifuged at 4°C. at 9250×g for 15 minutes to collect a supernatant. The supernatantwas repeatedly subjected to the treatment with phenol and the treatmentwith chloroform:isoamyl alcohol to obtain a supernatant aftercentrifugation. To the supernatant, an equal amount of 4 M lithiumchloride was added, followed by being stationarily left to stand at −70°C. for 1 hour.

After thawing at room temperature, the sample was centrifuged at 4° C.at 9250×g for 30 minutes to obtain a precipitate. The precipitate waswashed with 2 M lithium chloride once and with 80% ethanol once. Afterdrying, the precipitate was dissolved in 2 ml of adiethylpyrocarbonate-treated solution to give a sample of purified totalRNA. The obtained total RNA was 2.38 mg.

The all amount of the total RNA was applied to poly(A)⁺RNA purificationkit (produced by STRATAGENE CLONING SYSTEMS) using an oligo(dT)cellulose column, so that poly(A)⁺RNA molecules were purified to obtain42.5 μg of poly(A)⁺RNA.

Single strand cDNAs were synthesized from poly(A)⁺RNA obtained asdescribed above, by using reverse transcriptase Super Script II(produced by GIBCO BRL). In order to isolate raffinose synthase cDNAfrom an obtained cDNA mixture, amplification was performed in accordancewith the PCR method. As primers in PCR, single strand oligonucleotides(SEQ ID NOs: 6 to 22) shown in FIG. 7 were synthesized on the basis ofthe amino acid sequences of the peptide fragments of the raffinosesynthase originating from cucumber, determined in Example 2. In thesequences of the respective primers, R represents A or G, Y represents Cor T, M represents A or C, K represents G or T, D represents G, A, or T,H represents A, T, or C, B represents G, T, or C, N represents G, A, T,or C, and I represents inosine (base: hypoxanthine) respectively.

A DNA fragment-of about 540 base pairs was amplified when the primerswere combined such that the 5′-side primer was A (A1 (SEQ ID NO: 6), A2(SEQ ID NO: 7), A3 (SEQ ID NO: 8), A4 (SEQ ID NO: 9)) and the 3′-sideprimer was D′ (D′1 (SEQ ID NO: 21), D′2 (SEQ ID NO: 22)), or the 5′-sideprimer was C2 (SEQ ID NO: 14) and the 3′-side primer was B′1 (SEQ ID NO:18) or B′2 (SEQ ID NO: 19). The fragment was cloned into a plasmid pCRIIby using TA cloning kit (produced by INVITROGEN BV) to analyze itsnucleotide sequence. As a result, a nucleotide sequence coding for theamino acid sequences of Peptides 1, 2 was found inwardly between theprimer sequences at both terminals. Accordingly, it was found that theamplified fragment is a DNA fragment originating from the raffinosesynthase gene.

In order to specify the position of the cloned PCR-amplified DNAfragment on the raffinose synthase gene, 3′-RACE was performed by usingRACE kit (3′ Ampifinder RACE Kit, produced by CLONTACH).

PCR was performed by using the cDNA mixture as a template, C (C1 (SEQ IDNO: 13), C2 (SEQ ID NO: 14)) as a 5′-side primer, and a primer havingoligo(dT) and an anchor sequence as a 3′-side primer. Further, PCR wasperformed by using an amplified fragment thus obtained as a template, D(D1 (SEQ ID NO: 15), D2 (SEQ ID NO: 16)) located inwardly from C as a5′-side primer, and an oligo(dT)-anchor primer as a 3′-side primer. As aresult, a DNA fragment of about 2400 base pairs was amplified only whenPCR was performed by using, as the template, DNA amplified with C1 (SEQID NO: 13) or C2 (SEQ ID NO: 14) and the oligo(dT)-anchor primer, andusing D2 (SEQ ID NO: 16) and the oligo(dT)-anchor primer. Further, PCRwas performed by using C (C1 (SEQ ID NO: 13), C2 (SEQ ID NO: 14)) as the5′-side primer and the oligo(dT)-anchor primer as the 3′-side primer,and then PCR was performed by using the amplified fragment thus obtainedas a template, E (SEQ ID NO: 17) as a 5′-side primer, and theoligo(dT)-anchor primer as a 3′-side primer. As a result, a DNA fragmentof about 300 base pairs was amplified in any case.

Similarly, PCR was performed by using A (A1 (SEQ ID NO: 6), A2 (SEQ IDNO: 7), A3 (SEQ ID NO: 8), or A4 (SEQ ID NO: 9)) as a 5′-side primer,and the primer having oligo(dT) and the anchor sequence as a 3′-sideprimer. Further, PCR was performed by using an amplified fragment thusobtained as a template, and using B (B1 (SEQ ID NO: 10), B2 (SEQ ID NO:11), or B3 (SEQ ID NO: 12)) located inwardly from A as a 5′-side primer,and the same oligo(dT)-anchor primer as a 3′-side primer. As a result, aDNA fragment of about 2000 base pairs was obtained when the B2 primerwas used even in the case that any of the A primers was used. Thus theDNA fragment amplified by using the A2 and B2 primers was cloned. As aresult of nucleotide sequence analysis, the DNA fragment included, onthe 5′-side, the nucleotide sequence coding for the amino acid sequenceof Peptide fragment 1 used to prepare the primer. The DNA fragment alsoincluded, on the 3′-side, the poly(A) sequence and the nucleotidesequence corresponding to Peptide fragment 3 at a position locatedupstream therefrom.

In view of the result of PCR described above, it was found that Peptidefragments of the raffinose synthase are arranged from the N-terminalside in an order of 2, 1, 3, and the DNA fragment of about 540 basepairs previously obtained by PCR was a portion located near to the5′-terminal on the raffinose synthase gene. In order to screen a cDNAclone containing the entire length of the raffinose synthase gene, it isdesirable that DNA to be used as a probe can detect a portion near tothe 5′-terminal side. Accordingly, the obtained DNA fragment was used asa probe to perform screening for a cDNA library.

<2> Cloning of Entire Length of Coding Region of Raffinose Synthase cDNA

At first, a cDNA library was prepared as follows. Double strand cDNAswere synthesized from poly(A)⁺RNA (3.8 μg) obtained in the foregoingitem <1> by using Time Saver cDNA synthesis kit (produced by PharmaciaBiotech). Obtained cDNAs were incorporated into EcoRI restriction enzymecleavage site of λ phage vector, λMOSSlox (produced by Amersham)respectively, and then incorporated into the phage protein by usingGigapackII Gold packaging kit (produced by STRATAGENE CLONING SYSTEMS).Thus the cucumber cDNA library was prepared. This library had a titer of1.46×10⁷ pfu/μg vector.

Host cells of Escherichia coli ER1647 were infected with the phagescontained in the cucumber cDNA library in an amount corresponding to1.4×10⁵ pfu, and then the cells were spread over 14 agar plates eachhaving a diameter of 90 mm to give 1.0×10⁴ pfu per plate. The cells werecultivated at 37° C. for about 6.5 hours. After that, phage plaquesformed on the plates were transferred to nylon membranes (Hybond-N+,produced by Amersham).

Next, the nylon membranes were treated with alkali to denaturetransferred DNA, followed by neutralization and washing. After that, thenylon membranes were treated at 80° C. for 2 hours to fix DNA on themembranes.

Positive clones were screened on the obtained nylon membrane by usingthe DNA fragment of about 540 base pairs obtained in the foregoing item<1> as a probe. The DNA fragment of about 540 base pairs was digestedwith restriction enzyme EcoRI, followed by electrophoresis to excise andpurify only the insert of about 540 base pairs. The insert was labeledwith fluorescein by using DNA labeling and detection system (Gene Imageslabeling and detection system, produced by Amersham) to be used as theprobe. The nylon membranes were subjected to prehybridization at 60° C.for 30 minutes, and then the labeled probe was added to performhybridization at 60° C. for 16 hours. An antibody (alkalinephosphatase-labeled anti-fluorescein antibody) for detecting the labeledDNA was used after being diluted 50000 times. In this screening process,candidate strains for positive clones were obtained. The obtainedcandidate strains were further subjected to repeated screening twice inthe same manner as described above to obtain a purified positive clone.

Escherichia coli BM25.8 was infected with the positive clone, and it wascultivated on a selection medium containing carbenicillin. A plasmidvector λMOSSlox-CRS containing cDNA was excised therefrom. The insertedcDNA of the plasmid had a length of about 2.5 kb. Escherichia coli JM109was transformed with the plasmid. Plasmid DNA was prepared from atransformant, and was used as a sample for analyzing the nucleotidesequence.

The nucleotide sequence of the inserted cDNA was analyzed by using TaqDyeDeoxy Terminator Cycle Sequencing Kit (produced by Perkin-Elmer) inaccordance with the conventionally known method.

As a result, a nucleotide sequence comprising 2352 base pairs as shownin SEQ ID NO: 4 in Sequence Listing was revealed. The sequence includeda portion coincident with the nucleotide sequence of the DNA probe usedby the present inventors. An amino acid sequence translated from thenucleotide sequence is shown in SEQ ID NOs: 4 and 5. The amino acidsequence included portions coincident with Peptide 1 (amino acid numbersof 215 to 244 in SEQ ID NO: 5), Peptide 2 (amino acid numbers of 61 to79 in SEQ ID NO: 5), and Peptide 3 (amino acid numbers of 756 to 769 inSEQ ID NO: 5) of the raffinose synthase originating from cucumberobtained by the present inventors. Thus it was confirmed that the aminoacid sequence codes for the raffinose synthase.

The transformant, designated as AJ13263, of Escherichia coli JM109,which harbors the plasmid pMossloxCRS containing DNA coding for theraffinose synthase obtained as described above, has been internationallydeposited on the basis of the Budapest Treaty since November 19, 1996 inNational Institute of Bioscience and Human Technology of Agency ofIndustrial Science and Technology of Ministry of International Trade andIndustry (postal code: 305, 1-3 Higashi 1-chome, Tsukuba-shi,Ibaraki-ken, Japan), and awarded an accession number of FERM BP-5748.

EXAMPLE 4 Preparation of DNA Coding for Raffinose Synthase Originatingfrom Soybean

<1> Screening of Probe for Cloning Raffinose Synthase Gene Originatingfrom Soybean

Northern hybridization to soybean total RNA was carried out by using theentire length of the raffinose synthase gene originating from cucumberas probe. The probe was prepared by digesting the plasmid pMossloxCRSobtained in Example 3 with a restriction enzyme NotI, subjecting thedigest to agarose gel electrophoresis to isolate an inserted fragmentand labeling the isolated DNA fragment with α-P³² dCTP. As the totalRNA, 30 μg of total RNA prepared from immature seeds (5 to 6 weeks afterbloom) of soybean by the SDS-phenol method. After prehybridization for30 minutes, the probe was added to carry out hybridization at 42° C.overnight. Washing was carried out under a condition of 1×SSC, 0.1% SDS,and 60° C. The signal of the raffinose synthase originating fromcucumber as a control was observed, but no distinct signal was observedwith respect to the RNA originating from soybean. It was considered thatit was desirable to use a more conserved region rather than the entirelength of the cucumber gene.

<2> Isolation of Partial Fragment of Raffinose Synthase Gene ofArabidopsis thaliana

Pods 17 to 20 days after bloom (125 mg) of Arabidopsis thaliana wereground by a mortar with liquid nitrogen. To the ground material, 3 ml of2×CTAB (2% cetyltrimethylammonium bromide, 0.1 M Tris-HCl (pH 9.5), 1.4M NaCl, and 0.5% mercaptoethanol) was added, and allowed to diffusetherein, followed by agitation at 65° C. for 10 minutes. After themixture was transferred to Bluemax (50 ml; Falcon Tube), 3 ml ofchloroform:isoamyl alcohol (24:1(v:v)) was added thereto, followed bygentle agitation. An obtained mixture solution was centrifuged at 12000rpm for 10 minutes to collect a supernatant. The supernatant was againextracted with chloroform:isoamyl alcohol (24:1(v:v)) to obtain asupernatant after centrifugation at 10000 rpm for 25 minutes. To 1.8 mlof the supernatant, 1.5 ml of isoamyl alcohol was added and mixed toobtain a precipitate after centrifugation at 12000 rpm for 15 minutes at4° C. The precipitate was washed with 70% ethanol and dried, and thendissolved in 1 ml of TE buffer. To the solution, a quarter amount of 10M lithium chloride was added and mixed, and allowed to stand on ice for4 hours. After centrifugation at 12000 rpm for 15 minutes at 4° C., aprecipitate was washed with 2 M lithium chloride, and with 70% ethanol,dried and then dissolved in 100 μl of TE buffer. To the solution,phenol:chloroform (1:1(v/v) was added and agitated to an aqueous layerafter centrifugation at 12000 rpm for 15 minutes at 4° C. The aqueouslayer was subjected to ethanol precipitation, and an obtainedprecipitate was washed with 70% ethanol and dried, and then dissolved in10 μl of a diethylpyrocarbonate-treated solution to give a sample ofpurified total RNA. The obtained total RNA was 18.7 μg. Single strandcDNAs were synthesized from the total RNA by using reverse transcriptaseSuper Script II (produced by GIBCO BRL).

In order to amplify a partial fragment of the raffinose synthase genefrom an obtained cDNA mixture in accordance with the PCR method, primerswere synthesized. DNAs having homology with the raffinose synthase geneoriginating from cucumber were searched on GenBank, and primers weresynthesized as single strand oligonucleotides (SEQ ID NOs: 25 and 26) onthe basis of the conserved region. A DNA fragment of about 250 basepairs was amplified when PCR was carried out by using the primers andthe single strand cDNA as a template. The fragment was cloned into aplasmid pCRII by using TA cloning kit (produced by INVITROGEN BV) toanalyze its nucleotide sequence. As a result, a nucleotide sequenceshown in SEQ ID NO: 27 was obtained. It was considered that the partialcDNA fragment of the raffinose synthase originating from Arabidopsisthaliana, which had homology with the raffinose synthase originatingfrom cucumber, was obtained.

<3> Cloning of cDNA of Raffinose Synthase Originating from Soybean

Seeds 5 to 6 weeks after bloom (4.5 g) of soybean were ground by amortar with liquid nitrogen. From the ground material, 1.3 mg of totalRNA was prepared by SDS-phenol method. The total RNA was applied to anoligo(dT) cellulose column (poly(A)⁺RNA purification kit; produced bySTRATAGENE CLONING SYSTEMS) to isolate about 6 μg of poly(A)⁺RNA. Fromabout 2 μg of poly(A)⁺RNA obtained, double strand cDNAs were synthesizedusing Time Saver cDNA synthesis kit (produced by Pharmacia Biotech) witholigo dT primers. Obtained cDNAs were incorporated into EcoRIrestriction enzyme cleavage site of λ phage vector, λMOSSlox (producedby Amersham) respectively, and then incorporated into the phageparticles by using GigapackII Gold packaging kit (produced by STRATAGENECLONING SYSTEMS). Thus the soybean cDNA library was prepared. Thislibrary had a titer of 1.42×10⁷ pfu/pg vector.

Phages contained in the soybean cDNA library in an amount correspondingto 1.4×10⁵ pfu were transferred and fixed to nylon membrans (Hybond-N+,produced by Amersham) as in the cucumber cDNA library. With respect toeach plate, transfer to two membranes was carried out to prepare twosets. The obtained membranes were screened by using the partial cDNAfragment of the raffinose synthase originating from Arabidopsisthaliana. This DNA fragment was used as a probe by labelling withfluorescein by Gene Image labelling detect system (produced byAmersham). Hybridization and detection were carried out as in thecucumber cDNA library screening, except that washing of the membrane wascarried out at 1×SSC and 0.1% SDS for one on the sets and at 0.1×SSC and0.1% SDS for another. 15 of positive clone candidates were obtainedunder both conditions. As to the candidates, screening as describedabove was once repeated to obtain 5 purified clones.

Escherichia coli BM25.8 was infected with each of the positive clones,and a plasmid containing the cDNA was excised therefrom. Also,Escherichia coli JM109 was transformed with the plasmid, and plasmid DNAwas prepared from the transformant and used as a sample for sequenceanalysis. Sequence analysis was carried out in the same manner as in thecase of the raffinose synthase gene originating from cucumber. Based onthe sequence analysis, it was confirmed that one of 5 clones,pMOSSloxSRS contained the entire length of the raffinose synthase geneoriginating from soybean.

The insert fragment of pMOSSloxSRS had a nucleotide sequence comprising2780 base pairs as shown in SEQ ID NO: 23 in Sequence Listing, andencoded the raffinose synthase composed of 750 amino acids. The insertfragments of other clones were shorter than that of pMOSSloxSRS andlacked 5′ side of the raffinose synthase.

The transformant, designated as AJ13379, of Escherichia coli JM109,which harbors the plasmid pMOSSloxSRS containing a DNA fragmentcontaining DNA coding for the raffinose synthase obtained as describedabove, has been internationally deposited on the basis of the BudapestTreaty since Oct. 20, 1997 in National Institute of Bioscience and HumanTechnology of Agency of Industrial Science and Technology of Ministry ofInternational Trade and Industry (postal code: 305, 1-3 Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan), and awarded an accession number ofFERM BP-6149.

EXAMPLE 5 Chimeric Gene and Transformed Plant Containing DNA Coding forRaffinose Synthase

<1> Construction of Plasmid Containing Chimeric Gene

The DNA fragment coding for the raffinose synthase was introduced intoArabidopsis thaliana by using LBA4404 as Agrobacterium and pBI121(produced by CLONTECH) as a binary vector. pBI121 is a plasmidoriginating from pBIN19, which comprises nopaline synthase gene promoterconnected to neomycin phosphotransferase structural gene (NPTII),nopaline synthase gene terminator (Nos-ter), CaMV 35S promoter, GUS(β-glucuronidase) gene, and Nos-ter, and which has sequences forenabling transposition to plant, on both sides thereof. A SmaI site islocated downstream from CaMV 35S promoter. An insert inserted into thissite is expressed under the regulation of the promoter.

A fragment of the raffinose synthase gene originating from cucumberobtained in Example 3 was inserted into the binary vector pBI121. Theraffinose synthase gene was digested with DraI to prepare, by means ofagarose gel electrophoresis, a DNA fragment containing 1382nd to 2529thnucleotides in SEQ ID NO: 4 in Sequence Listing. This fragment wasligated into the SmaI site of pBI121. Escherichia coli HB101 wastransformed with the ligation reaction solution to obtain transformantstrains, and recombinant plasmids were prepared therefrom. Tworecombinant plasmids, in which the raffinose synthase DNA fragment wasreversely connected to CaMV 35S promoter (antisense), and the raffinosesynthase DNA fragment was connected to CaMV 35S promoter in the ordinarydirection (sense), were selected from the obtained recombinant plasmids.The two recombinant plasmids were designated as pBIcRS1 and pBIcRS9respectively.

Also, a plasmid containing a chimeric gene expressing the raffinosesynthase was constructed. pMOSSloxSRS containing the raffinose synthasegene was digested with NotI and a raffinose synthase gene fragment wasprepared by means of agarose gel electrophoresis. The NotI cleavagesites of the DNA fragment were filled in by the Taq polymerase reactionusing dNTP to obtain a SRS fragment having A bases protruded at 3′ side.On the other hand, pBI121 was digested with SmaI, a linear DNA waspurified by means of agarose gel electrophoresis, and pBI121/SmaI havingT bases added at 3′ side was obtained by the Taq polymerase reactionusing dTTP. After purification, the SRS fragment was ligated topBI121/SmaI. Escherichia coli HB101 was transformed with the ligationreaction solution. Plasmid DNAs were prepared from the obtainedtransformants and digested with each of restriction enzymes EcoRI,BamHI, XhoI or a combination thereof. Molecular weights of the obtainedfragments were determined by agarose gel electrophoresis to prepare aphysical map. Based on the prepared physical map, one in which theraffinose synthase gene was connected to the CaMV 35S promoter in theordinary direction, was selected from the recombinant plasmids anddesignated as pBIsRS1.

Each of the plasmids obtained as described above was introduced intoAgrobacterium LBA4404 by means of triparental mating of Escherichia coliHB101 containing the plasmids and Agrobacterium LBA4404.

<2> Transformation

Arabidopsis thaliana was transformed as follows. Seeds of Arabidopsisthaliana was subjected to a treatment for water absorption. After that,they were sterilized by treating them with 80% ethanol containing 1%Tween 20 for 5 minutes, and treating them with 10% sodium hypochloritesolution also containing 1% Tween 20 for 10 minutes, followed by washingfive times with sterilized water. The seeds were suspended in 1% lowmelting point agarose, and they were spread over an MS medium (MS basicmedium (Murashige and Skoog, Physiologia Plantrum, 15, 473-497 (1962)),B5 vitamin, 10 g/L sucrose, 0.5 g/L MES, pH 5.8). The seeds werecultivated at 22° C. for 1 week in a culture room to give a cyclecomprising light irradiation for 16 hours and darkness for 8 hours.Plants with seed leaves expanded were subjected to setting with rockwool. Cultivation was continued under the same condition. After about 3weeks, decapitation was performed when the plants caused bolting to havestems of heights of several centimeters. The plants were allowed to growuntil a state in which first flowers bloom on elongated branches 1 weekafter the decapitation.

Agrobacterium harboring the introduced recombinant plasmid containingthe raffinose synthase gene was precultivated in 2 ml of LB medium. Anobtained culture was inoculated into LB medium containing 50 mg/Lkanamycin and 25 mg/L streptomycin, followed by cultivation at 28° C.for about 1 day. Bacterial cells were collected at room temperature, andthey were suspended in a suspension medium for infiltration (½ MS salt,½ Gamborg B5 vitamin, 5% sucrose, 0.5 g/L MES, pH 5.7 (KOH), to which,immediately before the use, benzylaminopurine was added to give a finalconcentration of 0.044 μM, and Silwet L77 was added in an amount of 200μl per liter (final concentration: 0.02%)) so that OD₆₀₀ of an obtainedbacterial suspension was 0.8.

Flowers in bloom and fructification were removed from the plants to besubjected to infiltration. The rock wool was inverted upside down, andflowers which were not in fructification were immersed in the suspensionof Agrobacterium, followed by being placed in a desiccator so that thepressure was reduced to be 40 mmHG for 15 minutes. Seeds were harvestedafter 2 to 4 weeks. The harvested seeds were stored in a desiccator.

Next, transformants were selected on a selection medium. The seeds weresterilized in the same manner as described above, and they werecultivated on a selection medium (MS salt, Gamborg B5 vitamin, 1%sucrose, 0.5 g/L MES, pH 5.8, 0.8% agar, to which antibiotics forselection, i.e., carbenicillin (final concentration: 100 mg/L) andkanamycin (final concentration: 50 mg/L) were added after autoclaving))at 22° C. to select resistant plants. The resistant plants weretransferred to a fresh medium, and they were allowed to grow until trueleaves expanded. Seeds were harvested from the obtained plants.Selection was repeated in the same manner as described above, and thusT3 seeds were obtained. The T3 seeds were measured for the raffinosecontent in accordance with the method described above. Results are shownin Table 3.

TABLE 3 Plant Raffinose content (mg/g) Wild type 0.20 Transformant(pBIcRS1) 0.00 Transformant (pBIcRS9) 0.00 Transformant (pBIsRS1) 0.22

INDUSTRIAL APPLICABILITY

The present invention provides the purified raffinose synthase, theraffinose synthase gene, the chimeric gene comprising the raffinosesynthase gene and the regulatory region expressible in plants, and theplant to which the chimeric gene is introduced.

Raffinose can be efficiently synthesized from sucrose and galactinol byusing the raffinose synthase of the present invention. The content ofthe raffinose family oligosaccharides in plants can be changed byutilizing the raffinose synthase gene or the chimeric gene of thepresent invention.

1. An isolated raffinose synthase having an activity to produceraffinose from sucrose and galactinol and comprising SEQ ID NO:
 5. 2.The raffinose synthase of claim 1, which consists of SEQ ID NO:
 5. 3.The raffinose synthase of claim 1, which has the following properties:(1) action and substrate specificity: produces raffinose from sucroseand galactinol; (2) optimum pH: about 6 to 8; (3) optimum temperature:about 35 to 40° C.; (4) molecular weight: (i) about 75 kDa to 95 kDaestimated by gel filtration chromatography; (ii) about 90 kDa to 100 kDaestimated by polyacrylamide gel electrophoresis; and (iii) about 90 kDato 100 kDa estimated by SDS-polyacrylamide gel electrophoresis under areduced condition; and (5) inhibition: inhibited by iodoacetamide,N-ethylmaleimide, and myo-inositol.
 4. The raffinose synthase of claim3, which is isolated from cucumber.
 5. A method for producing raffinose,comprising the step of allowing the raffinose synthase as defined inclaim 1 to act on sucrose and galactinol to produce raffinose.
 6. Amethod for producing raffinose, comprising the step of allowing theraffinose synthase as defined in claim 2 to act on sucrose andgalactinol to produce raffinose.
 7. A method for producing raffinose,comprising the step of allowing the raffinose synthase as defined inclaim 3 to act on sucrose and galactinol to produce raffinose.
 8. Amethod for producing raffinose, comprising the step of allowing theraffinose synthase as defined in claim 4 to act on sucrose andgalactinol to produce raffinose.